Diagnosis of liver pathology through assessment of protein glycosylation

ABSTRACT

Methods for diagnosing pathology of the liver in a subject suspected of having such pathology are disclosed. The methods comprise quantifiably detecting lectin binding on proteins in biological fluids, and comparing the detected lectin binding with reference values for the binding of lectin of such proteins in healthy or disease states.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/443,326, filedApr. 10, 2012, which is a continuation of U.S. Ser. No. 12/853,486 (nowU.S. Pat. No. 8,183,000), which is a continuation of U.S. Ser. No.11/418,598 (now U.S. Pat. No. 7,776,550), which claims benefit to U.S.Provisional Application No. 60/677,941, filed May 5, 2005, the entirecontents of each of which are incorporated by reference herein.

GOVERNMENT SUPPORT

Research leading to the disclosed inventions was funded, in part, withfunds from the NCI under grants R33CA94340 and U01 CA84951. Accordingly,the United States government may have certain rights in the inventionsdescribed herein.

FIELD OF THE INVENTION

The invention relates generally to the field of immunodiagnostics. Morespecifically, the invention relates to methods and kits for rapid andaccurate diagnosis of liver diseases such as hepatocellular carcinoma,hepatitis, and cirrhosis via the detection of specific fucosylatedglycoproteins identified as being associated with liver pathologies.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety.

The liver is the largest gland in the body, and plays a vital role in,among other things, digestion, metabolism of carbohydrates, lipids, andproteins, storage of vitamins, minerals, and carbohydrates, productionof blood clotting factors, destruction of bacteria in the blood, anddetoxification of the body from endogenous and exogenous substances.Given the liver's broad spectrum of functions, diseases and pathologiesof the liver can have wide-ranging systemic effects on the body.

One common liver pathology is hepatocellular carcinoma (HCC). HCC ranksfifth of the most common cancers in the world, and is the third leadingcause of cancer death (El-Serag H et al. (2001) Hepatology 33:62-5; and,Block T et al. (2003) Oncogene 22:5093-107). The primary etiology forHCC is viral infection, particularly, infection with hepatitis B virus(HBV) and hepatitis C virus (HCV) (Brechot C (1996) Baillieres Clin.Gastroenterol. 10:335-73). HCC can lead to liver cirrhosis. In addition,cirrhosis is a risk factor for HCC (Ikeda K et al. (1993) Hepatology18:47-53).

Liver cirrhosis is characterized by, among other things, extensivefibrosis, hepatocyte necrosis, collapse of the supporting reticulinnetwork, and extensive deposition of connective tissue. There aremultiple etiologies for liver cirrhosis, including viral hepatitis,alcohol abuse, genetics (e.g., Wilson's Disease), venous thromboses inBudd-Chiari Syndrome, and autoimmunity (e.g., Primary BiliaryCirrhosis). Cirrhosis of the liver is irreversible, and if notcontrolled, can lead to liver failure. In fact, liver cirrhosis is aleading cause of death among adults in the United States, and throughoutthe world.

It is important that liver diseases such as HCC be detected early inorder to provide the patient with the full range of therapeutic optionsand ultimately improve patient prognosis (Hoofnagle, J H et al. (1997)N. Engl. J. Med. 336:347-56). Furthermore, it is equally important thatconditions that predispose to HCC and other liver diseases, for example,cirrhosis, and HBV and HCV infection, be detected early for effectivetreatment, and for the prevention of the onset of HCC. Unfortunately,many liver diseases, including HBV and HCV infection, can beasymptomatic for many years

In general, liver diseases are diagnosed and monitored by serologictesting, and liver function testing, as well as by physical examinationof the patient. In addition, as there is an apparent correlation betweenelevated expression of alpha-fetoprotein (AFP) and the presence of HCC,screening for AFP is often carried out as a matter of course in cases ofsuspected liver disease (Buamah P K et al. (1984) Clin. Chim. Acta139:313-6). However, AFP suffers from several major drawbacks insofar asit can be expressed in the absence of disease, leading to false positivediagnoses, and it is not found to be elevated in up to 50% of livercancer cases, leading to false negative diagnoses (Nguyen M H et al.(2002) Hepatology 36:410-7). Moreover, the predictive value of AFPsubstantially diminishes with respect to its capacity to identify earlystage HCC (Oka H et al. (1994) Hepatology 19:61-7; Pateron D et al.(1994) J. Hepatol. 20:65-72; and, Zoli M et al. (1996) Cancer78:977-83).

Thus, more rapid, accurate, and reliable means for the diagnosis ofliver diseases that are minimally invasive to the patient, and can bereadily and cost-effectively administered to all patients suspected ofhaving liver disease are needed. In addition, there is a need fordiagnostic tests that can detect the presence of disease in itsincipient or early stages to facilitate effective prophylactic treatmentof the patient.

SUMMARY OF THE INVENTION

The present invention features methods for diagnosing pathologies of theliver or biliary system. Generally, the methods comprise obtaining atest sample such as a biological fluid from a subject suspected ofhaving a pathology of the liver or biliary system, quantifiablydetecting glycosylation on proteins in the sample, and then comparingthe detected glycosylation with reference values for glycosylation ofsuch proteins. The reference values are established from subjects withno liver or biliary system pathology and from subjects with known liverpathologies. Either or both reference values can be compared with thedetected glycosylation levels, and the comparison will reveal thepresence or absence of the pathology of the liver or biliary system.

The inventive methods can be applied to detect any liver pathology, butare preferably applicable to detect hepatocellular carcinoma, hepatitis,cirrhosis, or combinations thereof. The preferred glycosylation that isdetected is fucosylation. Any fucosylated protein that is now or isidentified in the future as being associated with liver or biliarysystem pathology can be used as the target analyte. Non-limitingexamples of fucosylated proteins that have been identified includeGP-73, Hemopexin, HBsAg, hepatitis B viral particle,alpha-acid-glycoprotein, alpha-1-antichymotrypsin,alpha-1-antichymotrypsin His-Pro-less, alpha-1-antitrypsin,Serotransferrin, Ceruloplasmin, alpha-2-macroglobulin,alpha-2-HS-glycoprotein, alpha-fetoprotein, Haptoglobin, Fibrinogengamma chain precursor, immunoglobulin (including IgG, IgA, IgM, IgD,IgE, and the like), APO-D, Kininogen, Histidine rich glycoprotein,Complement factor 1 precursor, complement factor I heavy chain,complement factor I light chain, Complement C1s, Complement factor Bprecursor, complement factor B Ba fragment, Complement factor B Bbfragment, Complement C3 precursor, Complement C3 beta chain, ComplementC3 alpha chain, C3a anaphylatoxin, Complement, C3b alpha′ chain,Complement C3c fragment, Complement C3dg fragment, Complement C3gfragment, Complement C3d fragment, Complement C3f fragment, ComplementC5, Complement C5 beta chain, Complement C5 alpha chain, C5aanaphylatoxin, Complement C5 alpha′ chain, Complement C7, alpha-1 Bglycoprotein, B-2-glycoprotein, Vitamin D-binding protein,Inter-alpha-trypsin inhibitor heavy chain H2, Alpha-1B-glycoprotein,Angiotensinogen precursor, Angiotensin-1, Angiotensin-2, Angiotensin-3,GARP protein, beta-2-glycoprotein, Clusterin (Apo J), Integrin alpha-8precursor glycoprotein, Integrin alpha-8 heavy chain, Integrin alpha-8light chain, hepatitis C viral particle, elf-5, kininogen,HSP33-homolog, lysyl endopeptidase and Leucine-rich repeat-containingprotein 32 precursor.

The detection can proceed via any assay suitable in the art. Thedetection reagent can directly label the glycosyl moieties, for example,via carbohydrate specific chemicals or dyes, or via labeled lectins,labeled carbohydrate binding proteins, or labeled antibodies. Thedetection reagent can be a secondary reagent, for example, by firstcapturing the target analyte and then contacting the capturereagent-target complex with a labeled secondary reagent. Detection canproceed by separating glycosyl moieties from the proteins prior to thequantifiable detection of glycosylation. Detection can proceed byseparating glycoproteins from the test sample prior to the quantifiabledetection of glycosylation.

The invention also feature novel methods for detecting glycosylatedproteins in a sample. Such methods comprise contacting a sample with alectin and detecting the lectin-glycosylated protein complex. Theglycosylated proteins can be fucosylated proteins. The lectin can bedirectly coupled to a detectable moiety, or detection can proceed via asecondary reagent that specifically binds to the lectin, such as ananti-lectin antibody. The methods can comprise first contacting thesample with an antibody to capture target glycosylated proteins in thesample, for example, an antibody specific for the glycoproteinsexemplified herein.

Also featured in the present invention are kits for diagnosingpathologies of the liver or biliary system. The kits comprise a reagentthat specifically binds to glycosyl moieties, preferably fucosylmoieties. The reagent can be labeled with a detectable moiety, or can bea chemical that specifically labels glycosyl moieties, preferablyfucosyl moieties. If the reagent supplied with the kits is not coupledto a detectable moiety, the kit can further comprise a detection reagentthat specifically recognizes the reagent-glycoprotein complex, thedetection reagent being coupled to a detectable moiety. The kits furtherinclude instructions for using the kit in a method to diagnose apathology of the liver or biliary system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of glycoforms. Normally, liver cells produceglycoproteins that contain the carbohydrate structure shown in panel A.This is referred to as a bi-antennary glycan (A2G2). In HCC, the livercells attach a fucose residue to the glycan chain resulting in afucosylated glycoprotein (glycoform, referred to with the Fc prefix).Examination of all proteins having a specific carbohydrate chain isreferred to as targeted glycoproteomics. Abbreviations in the figure areas follows: N-acetylglucosamine (GlcNAc); mannose (Man); galactose;(Gal); sialic acid (NeuNAc); Fucose (Fuc).

FIG. 2 shows the level of the FcA2G2 glycan in people as a function oftime. (A) The level of the FcA2G2 structure in a patient either before(upper panel) or after (lower panel) the diagnosis of cancer. As thisfigure shows, the level of the FcA2G2 structure increases from 7.23% ofthe total glycan pool to over 13% of the total glycan pool after thediagnosis of cancer. (B) Levels of the FcA2G2 structure in 8 individualseither before or after the diagnosis of cancer. On the graph, the Y axisis the percentage of FcA2G2 structure in each individual as a functionof total released glycan. The X axis is the sample number.

FIG. 3 shows the lectin ELISA design utilized for Fc-AFP andFc-Kininogen. Periodate oxidized antibody was used as the captureantibody and the level of fucosylated protein determined by an alkalinephosphate conjugated lectin (LcH) using a colorimetric substrate(5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT)).

FIG. 4 shows a lectin analysis of human immunoglobulins in patients withvarying degrees of liver disease. Lectin ELISAs were performed asdescribed in FIG. 3 and the examples, except the lectin AAL (Aleuriaaurantia lectin) was used. 5 μl of human serum was used in the assay.This figure shows an increase in the level of fucosylated immunoglobulinwith increasing fibrosis. The differences between the cirrhotic groupand the healthy, stage 1&2 groups is statistically significant with ap<0.001 as determined by Student's t-test.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

DEFINITIONS

The following abbreviations may be used in the specification andexamples: HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitisC virus, HDV, hepatitis D virus; HEV, hepatitis E virus; HFV, hepatitisF virus; HGV, hepatitis G virus; AFP, alpha-fetoprotein; HCC,hepatocellular carcinoma; HPLC, high performance liquid chromatography;Fc, fucosylated.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

“Pathology” means any condition that is a deviation from the normal orhealthy state.

The term “biliary system” refers to the organs and duct system thatcreate, transport, store, and release bile into the small intestine. Theterm encompasses the liver, gallbladder, and bile ducts: the cysticduct, hepatic duct, common hepatic duct, common bile duct, andpancreatic duct.

Diseases and pathologies of the liver or biliary system abound.Non-limiting examples of liver or biliary diseases include AlagilleSyndrome, Alcoholic Liver Disease, Alpha-1-antityrpsin Deficiency,Autoimmune Hepatitis, Budd-Chiari Syndrome, Biliary Atresia, BylerDisease, Caroli Disease, Cholangiocarcinoma, Crigler-Najjar Syndrome,Drug- or Alcohol-induced Hepatitis, Dubin-Johnson Syndrome, FattyLiver/Steatosis, Gilbert Syndrome, Hemangioma, Hemohromatosis, HepatitisVirus A, B, C, D, E, and G, Hepatocellular Carcinoma,Hyperbilirubinemia, Primary Biliary Cirrhosis, Protoporphyria, RotorSyndrome, Sclerosing Cholangitis, and Wilson Disease.

A common liver pathology is cirrhosis. Cirrhosis of the liver ischaracterized by, among other things, widespread nodule formation in theliver, extensive fibrosis, hepatocyte necrosis, collapse of thesupporting reticulin network, extensive deposition of connective tissue,diminished blood flow through the liver, decreased bilirubin secretion,jaundice, and the disruption of normal liver biochemical functions.There are multiple etiologies for liver cirrhosis. More common causesinclude damage done to the liver by ingestion of alcohol, drugs, ortoxins, especially as in the case of alcoholic liver disease. Cirrhosismay also be virally induced by, for example, different strains of thehepatitis virus, inherited (e.g., Wilson's Disease, andhemochromatosis), induced by chronic disease or disruption of the bileducts, by parasitic infections (e.g., schistosomiasis), by excess ironabsorption, by autoimmunity (e.g., Primary Biliary Cirrhosis), or byliver inflammation resulting from fatty liver disease, among others.

“Hepatitis” refers to any clinically significant inflammation of theliver or biliary system, regardless of etiology. “Acute hepatitis”refers to any short term (less than six months) or initial-stage liverinflammation, such as the initial stages of hepatitis virus infection.“Chronic hepatitis” refers to any inflammation of the liver persistingsix months or longer. “Infectious hepatitis” refers to any inflammationof the liver that can be transmitted to others. Typically, infectioushepatitis is caused by a microorganism such as a virus (e.g., HAV, HBV,HCV, HDV, HEV, HFV, HGV, cytomegalovirus, Epstein-Barr virus, herpessimplex virus (HSV), and Varicella-Zoster virus, etc.), bacteria,protozoan, or yeast. “Non-infectious hepatitis” refers to anyinflammation of the liver that cannot be transmitted to others, such asalcoholic hepatitis, autoimmune hepatitis, toxic/drug induced hepatitis,and granulomatus hepatitis, and the like.

“Etiology” means the cause or origin of a disease, disorder, orpathology.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asantibody fragments (e.g., Fab, Fab′, F(ab′)₂ and F_(v)), including theproducts of a Fab or other immunoglobulin expression library. Withrespect to antibodies, the term, “immunologically specific” or“specific” refers to antibodies that bind to one or more epitopes of aprotein of interest, but which do not substantially recognize and bindother molecules in a sample containing a mixed population of antigenicbiological molecules. Screening assays to determine binding specificityof an antibody are well known and routinely practiced in the art. For acomprehensive discussion of such assays, see Harlow et al. (Eds.),ANTIBODIES A LABORATORY MANUAL; Cold Spring Harbor Laboratory; ColdSpring Harbor, N.Y. (1988), Chapter 6.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from natural posttranslational processesor may be made by synthetic methods.

“Post-translational modification” refers to any chemical modification ofa polypeptide after it is produced. Commonly, a post-translationalmodification involves attaching at least one moiety to the polypeptidechain, however, post-translations modification can be cleavage of thepolypeptide chain, proteolytic processing, the formation of disulfidebonds, and the like. Non-limiting examples of post-translationalmodifications include, glycosylation, phosphorylation, acylation,acetylation, methylation, sulfonation, prenylation, isoprenylation,ubiquitination, biotinylation, formylation, citrullination,myristolation, ribosylation, sumoylation, gamma carboxylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, GPI anchor formation,hydroxylation, iodination, methylation, oxidation, proteolyticprocessing, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andthe like. See, for instance, Proteins—Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al, (1990) Analysis for Protein Modifications andNonprotein Cofactors, Methods Enzymol. 182:626-46 and Rattan et al.(1992) Protein Synthesis: Posttranslational Modifications and Aging,Ann. NY Acad. Sci. 663:48-62.

As used herein, “glycosylation” refers to the chemical attachment of atleast one saccharide moiety to a molecule such as a polypeptide.Glycosylation can be n-linked or o-linked.

“Fucosylation” refers to the chemical attachment of at least one fucosemoiety to a molecule such as a protein. A “fucosylated” polypeptide is apolypeptide with at least one fucose moiety attached.

“Core glycosylation” refers to the addition of glycosyl moieties to thecore N-acetylglucosamine. “Core fucosylation” refers to the addition ofa fucose residue to the core N-acetylglucosamine. All N-linked glycanstructures have a common structure, referred to as the core, containingthree mannose, and two N-acetylglucosamine residues.

A “glycoform” refers to a group of proteins having the identical aminoacid sequence but having different carbohydrate moieties.

It has been discovered in accordance with the present invention thatthere is a correlation with the incidence of liver disease such ashepatocellular carcinoma, hepatitis virus infection, and cirrhosis andan increase in the level of core fucosylation. Analysis of the serumproteome from patients with liver disease revealed over fiftyglycoproteins that demonstrate increased fucosylation relative tohealthy controls, thereby revealing that increased protein fucosylationis indicative of liver disease state. Accordingly, in one aspect, theinvention features methods to diagnose a pathology of the liver orbiliary system in a patient suspected of having a pathology of the liveror biliary system. Such methods comprise obtaining a test sample from apatient suspected of having a pathology of the liver, and quantifiablydetecting post-translational modifications on proteins in the testsample, wherein modulated levels of the post-translational modificationsrelative to reference values for post-translational modifications forsuch proteins indicates the presence or absence of a pathology of theliver.

A test sample can be obtained from any location in a patient in whichpost-translationally modified proteins indicative of a condition of theliver are likely to be found. For example, a test sample can be obtainedfrom biological fluids such as tears, saliva, mucous, whole blood,serum, plasma, urine, bile, and the like. A test sample could also beobtained from specific cells or tissue, or from any secretions orexudate. For example, a biopsy of cells or tissues from the liver orbiliary system can serve as a test sample. Preferably, the test sampleis obtained from peripheral blood.

In one preferred embodiment, detection of the post-translationalmodification can be carried out by detecting a polypeptide-moietycomplex. In another preferred embodiment, detection of thepost-translational modification can be carried out by separating thepolypeptide and moiety, and detecting the moiety.

Detection of the polypeptide-moiety complex can be carried out using areagent that specifically recognizes the moiety, the particular class ofmoiety, or the moiety as a complex with the polypeptide. Suitabledetection reagents will be apparent to those of skill in the art,non-limiting examples of which are described below. The reagent cancomprise multiple molecules each having specificity for a differenttarget moiety, thereby resulting in multiple reagent-targetinteractions.

Antibodies can be used as the reagent. Any antibody that specificallybinds to the target moiety of interest can be used in the presentinvention. Monoclonal and/or polyclonal antibodies can be used, fromwhatever source produced, as can recombinant antibodies such as singlechain antibodies and phage-displayed antibodies, as well as chimeric andhumanized antibodies. Antigen binding fragments of antibodies such asthe Fab or Fv can also be used.

In some embodiments, the antibodies specifically recognizeglycoproteins. Preferably, the antibodies specifically recognizecarbohydrate moieties, including mono- and poly-saccharides. Morepreferably, the antibodies specifically recognize fucose moieties.Antibodies capable of specifically recognizing fucose have beendescribed. See, e.g., Roy S S et al. (2002) Ann. Bot. 89:293-9; and,Srikrishna G et al. (1998) Glycobiology 8:799-811. In the alternative,antibodies can also be raised to various moieties, including fucose, andused in the invention. Methods for raising and purifying antibodies arewell known in the art. In addition, monoclonal antibodies can beprepared by any number of techniques that are known in the art,including the technique originally developed by Kohler and Milstein(1975) Nature 256:495-497.

Other proteins that have carbohydrate recognition domains can also beused as the reagent in the present invention. Proteins havingcarbohydrate recognition domains have been described, see, e.g., BouyainS et al. (2002) J. Biol. Chem. 277:22566-72 (Drosophila melanogasterprotein CG2958 that recognizes fucose).

In particularly preferred embodiments, lectins are used as the reagent.The lectins can be obtained from any organism, including plants,animals, yeast, bacteria, protozoans, and the like. Purified lectins arecommercially available, see, e.g., Sigma-Aldrich catalog (St. Louis,Mo.). Lectins can also be isolated from their naturally occurringsource, or recombinantly expressed and purified, by means that arewell-known to those of skill in the art. The lectin can, but need not bespecific for a particular carbohydrate moiety. Fucose-specific lectinshave been described. See, e.g., Mansour M H et al. (2005) Immunobiology.210:335-48; Amano K et al. (2003) Biosci. Biotechnol. Biochem.67:2277-9; Loris R et al. (2003) J. Mol. Biol. 331:861-70; and, Ishida Het al. (2002) Biosci. Biotechnol. Biochem. 66:1002-8. It is contemplatedthat future-identified lectins are suitable for use in the presentinvention.

Proteins with lectin-like domains are also suitable for use in thepresent invention. Proteins with lectin-like domains are known in theart. See, e.g., Drickamer K (1999) Curr. Opin. Struct. Biol. 9:585-90.

Nucleic acid-based alternatives to lectins can also be used. Suchreagents, termed aptamers, take advantage of the huge conformationalflexibility of single stranded nucleic acids. From large pools ofrandomized short nucleic acids, individual molecules with highaffinities for numerous non-nucleic acid ligands have been isolated byiterative selection. Advantages of the glycan-binding “lectamer”reagents are that leached DNAs are unlikely to confuse or interfere withdownstream analyses. Lectamers can function under uniform bindingconditions (pH, ionic strength). Synthetic nucleic acids can be preparedin various derivatized forms (e.g., terminally biotinylated). Targetglycans are not limited by existing lectin specificities, substantiallyexpanding existing fractionation and analytical capabilities.

Any polypeptide on which post-translational modifications are modulatedupon the onset or progression of a pathology of the liver can be used inthe inventive diagnostic assays. Non-limiting examples of suchpolypeptides that have been characterized thus far include GP-73,Hemopexin, HBsAg, hepatitis B viral particle, alpha-acid-glycoprotein,alpha-1-antichymotrypsin, alpha-1-antichymotrypsin His-Pro-less,alpha-1-antitrypsin, Serotransferrin, Ceruloplasmin,alpha-2-macroglobulin, alpha-2-HS-glycoprotein, alpha-fetoprotein,Haptoglobin, Fibrinogen gamma chain precursor, immunoglobulin (includingIgG, IgA, IgM, IgD, IgE, and the like), APO-D, Kininogen, Histidine richglycoprotein, Complement factor 1 precursor, complement factor I heavychain, complement factor I light chain, Complement C1s, Complementfactor B precursor, complement factor B Ba fragment, Complement factor BBb fragment, Complement C3 precursor, Complement C3 beta chain,Complement C3 alpha chain, C3a anaphylatoxin, Complement, C3b alpha′chain, Complement C3c fragment, Complement C3dg fragment, Complement C3gfragment, Complement C3d fragment, Complement C3f fragment, ComplementC5, Complement C5 beta chain, Complement C5 alpha chain, C5aanaphylatoxin, Complement C5 alpha′ chain, Complement C7, alpha-1 Bglycoprotein, B-2-glycoprotein, Vitamin D-binding protein,Inter-alpha-trypsin inhibitor heavy chain H2, Alpha-1B-glycoprotein,Angiotensinogen precursor, Angiotensin-1, Angiotensin-2, Angiotensin-3,GARP protein, beta-2-glycoprotein, Clusterin (Apo J), Integrin alpha-8precursor glycoprotein, Integrin alpha-8 heavy chain, Integrin alpha-8light chain, hepatitis C viral particle, elf-5, kininogen,HSP33-homolog, lysyl endopeptidase and Leucine-rich repeat-containingprotein 32 precursor. It is contemplated that additionalpost-translationally modified proteins that are found to be correlatedwith a liver pathology can be used in the inventive methods.

The reagent can be directly labeled with a detectable moiety. In thealternative, a secondary reagent that specifically recognizes theprimary reagent, which is labeled with a detectable moiety is used. Thesecondary reagent can be any molecule, and is preferably an antibody.The secondary reagent is labeled with a detectable moiety. Detectablemoieties contemplated for use in the invention include, but are notlimited to, radioisotopes, fluorescent dyes such as fluorescein,phyocoerythrin, Cy-3, Cy5, allophycocyanin, DAPI, Texas red, rhodamine,Oregon green, lucifer yellow, and the like, green fluorescent protein,red fluorescent protein, Cyan Fluorescent Protein, Yellow FluorescentProtein, Cerianthus Orange Fluorescent Protein, alkaline phosphatase,β-lactamase, chloramphenicol acetyltransferase, adenosine deaminase,aminoglycoside phosphotransferase (neo^(r), G418^(r)) dihydrofolatereductase, hygromycin-B-phosphotransferase, thymidine kinase, lacZ(encoding α-galactosidase), and xanthine guaninephosphoribosyltransferase, Beta-Glucuronidase, Placental AlkalinePhosphatase, Secreted Embryonic Alkaline Phosphatase, or Firefly orBacterial Luciferase. Enzyme tags are used with their cognate substrate.As with other standard procedures associated with the practice of theinvention, skilled artisans will be aware of additional labels that canbe used. In some embodiments, the reagent or secondary reagent arecoupled to biotin, and contacted with avidin or strepatvidin having adetectable moiety tag.

In some embodiments, the moiety attached to the polypeptide bypost-translational modification can be directly labeled and detected,thereby obviating the need for a labeled reagent that specificallyrecognizes a particular moiety as well as any need for a labeledsecondary reagent. In some embodiments, the moiety attached to thepolypeptide by post-translational modification can be separated from thepolypeptide and directly labeled and detected. For example, and not byway of limitation, carbohydrates and carbohydrate moieties can bedirectly labeled using various methods that are known in the art.Carbohydrate and carbohydrate moiety labeling kits are commerciallyavailable. Carbohydrate and carbohydrate moieties can also bebiotinlyated and labeled with avidin- or streptavidin-conjugateddetectable moieties, such as those described herein. Non-limitingexamples of reagents that can directly label oligosaccharides include2-aminobenzamide and 2-aminobenzoic acid.

Post-translationally attached moieties can be separated from apolypeptide by any means suitable in the art, including chemically, forexample by treatment with hydrazine or acids such as hydrofluoric acidor trifluoromethanesulfonic acid, enzymatically, for example by bytreatment with N-glycosidase such as PNGase F, O-glycosidase,endoglycosidases, or exoglycosidases, or by physical means. Commerciallyavailable kits are available for removing post-translationalmodifications, including deglycosylation. Chemical bases such ashydrazine, or chemical reagents that lead to beta-elination reactionscan also be used in deglysosylation reactions. Other techniques andreagents will be appreciated by those of skill in the art, and arecontemplated to be within the scope of the present invention.

In some embodiments, the separated moieties are purified prior tolabeling or detection. Solid or liquid phase extraction techniques,which are known in the art, can be used to purify the separated moietiesfor further analysis.

Reference values can be those established for a particular liverpathology, or those established for healthy subjects, or both. Inaddition, the present invention contemplates that screening of testsamples using the inventive methods will reveal additionalpost-translationally modified proteins, and the particular type andlevel of post-translational modification of said proteins, whichcorrelate with disease state or healthy state. Using this information,these identified proteins can serve as additional reference valuesagainst which test samples can be compared.

A variety of assay formats can be used to carry out the inventivemethods, and to quantitatively detect post-translational modificationsof proteins. Immunoassays are one preferred assay, and include but arenot limited to ELISA, radioimmunoassays, competition assays, Westernblotting, bead agglomeration assays, lateral flow immunoassays,immunochromatographic test strips, dipsticks, migratory formatimmunoassays, and the like. Other suitable immunoassays will be known tothose of relevant skill in the art. Microscopy can also be used. In someembodiments, chromatography is the preferred assay. High performanceliquid chromatography (HPLC) is particularly preferred. In someembodiments, mass spectroscopy is the preferred assay. In someembodiments, gel electrophoresis coupled with densitometry is used asthe assay.

The general format of the assays involve contacting the reagent with atest sample containing the analytes of interest, namely thepost-translationally modified proteins, which may be distinguished fromother components found in the sample. Following interaction of theanalyte with the reagent, the system can be washed and then directlydetected or detected by means of a secondary reagent as exemplifiedherein.

In some preferred embodiments, the reagent is immobilized on a solidsupport. In other preferred embodiments, the test sample, or moleculesseparated or purified from the test sample, such as post-translationallymodified polypeptides, are immobilized on a solid support. Techniquesfor purification of biomolecules from samples such as cells, tissues, orbiological fluid are well known in the art. The technique chosen mayvary with the tissue or sample being examined, but it is well within theskill of the art to match the appropriate purification procedure withthe test sample source.

Examples of suitable solid supports include, but are not limited to,glass, plastic, metal, latex, rubber, ceramic, polymers such aspolypropylene, polyvinylidene difluoride, polyethylene, polystyrene, andpolyacrylamide, dextran, cellulose, nitrocellulose, pvdf, nylon,amylase, and the like. A solid support can be flat, concave, or convex,spherical, cylindrical, and the like, and can be particles, beads,membranes, strands, precipitates, gels, sheets, containers, wells,capillaries, films, plates, slides, and the like. The solid support canbe magnetic, or a column.

As the various post-translationally modifications associated with thepresence of a liver pathology identified to date can be present atdetectable levels within normal subjects (those without a liverpathology), it may be necessary to quantitatively measure the levels ofeach marker being analyzed in the diagnostic assay. In such cases,modulation of levels of the modifications relative to standards/controlswill be indicative of the presence of a liver pathology. Normalexpression levels of the various modifications can be empiricallydetermined according to any of various techniques that are known in theart. The normal expression levels can serve as a standard against whichthe expression levels in suspected liver pathology patients can becompared. Significant deviation (positive or negative) over expectednormal expression levels of liver pathology-associated modifications isindicative of the presence of a liver pathology in the patient.Similarly, the expression levels observed in confirmed liver pathologypatients can also serve as a standard against which the expressionlevels in suspected liver pathology patients can be compared. Similarlevels of expression of the liver pathology-associated markers betweenthe known patient and suspected patient is indicative of the presence ofa pathology in the patient. In such cases, it is expected that theexpression level of the modifications in both the known and suspectedsamples will significantly deviate from the level of expression presentin healthy subjects.

The present methods have applicability to diagnose a liver pathology inany animal. Preferably, the methods are utilized in mammals such asdogs, cats, horses, cows, pigs, rabbits, donkeys, sheep, mice, and rats.Most preferably, the methods are utilized in humans.

Also featured in the present invention are devices to diagnose apathology of the liver or biliary system in a patient suspected ofhaving a pathology of the liver. The devices comprise a reagent specificfor a post-translationally attached moiety associated with the presenceof a liver pathology, which is preferably coupled to a solid support.The devices are capable of use in any assay, particularly thosedescribed and exemplified herein, wherein the assay can quantifiablydetect the presence of the moiety associated with a liver pathology, andwherein modulated levels of the expression of the moiety relative to astandard indicates the presence of a pathology of the liver.

The reagents for use in the devices can comprise a single molecule thatcan form a complex with a single target, or multiple targets, forexample, a multimeric fusion protein with multiple binding sites fordifferent targets. The reagent can comprise multiple molecules eachhaving specificity for a different target. In preferred embodiments, thereagent is comprised of proteins. In some preferred embodiments, thereagent is of antibodies, and any antibody that specifically binds tothe target marker of interest can be used in the devices. In somepreferred embodiments, the reagent is comprised of carbohydraterecognition domains. In highly preferred embodiments, the reagent iscomprised of lectins or proteins with lectin domains, as exemplifiedherein.

The solid support to which the reagent is coupled can be any solidsupport described herein. The reagent can be immobilized on the solidsupport by any means suitable in the art, such as adsorption,non-covalent interactions such as hydrophobic interactions, hydrophilicinteractions, van der Waals interactions, hydrogen bonding, and ionicinteractions, electrostatic interactions, covalent bonds, or by use of acoupling agent. Coupling agents include glutaraldehyde, formaldehyde,hexamethylene diisocyanate, hexamethylene diisothiocyanate,N,N′-polymethylene bisiodoacetamide, N,N′-ethylene bismaleimide,ethylene glycol bissuccinimidyl succinate, bisdiazobenzidine,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, succinimidyl3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(N-maleimidometyl)cyclohexane-1-carboxylate (SMCC),N-sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate,N-succinimidyl (4-iodoacetyl)-aminobenzoate, N-succinimidyl4-(1-maleimidophenyl)butyrate,N-(epsilon-maleimidocaproyloxy)succinimide (EMCS), iminothiolane,S-acetylmercaptosuccinic anhydride,methyl-3-(4′-dithiopyridyl)propionimidate,methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate,N-succinimidyl-S-acetylmercaptoacetate, avidin, streptavidin, biotin,Staphylococcous aureus protein A, and the like.

Sites on the solid support not coupled with the capture reagent can beblocked to prevent non-specific binding of marker molecules to the solidsupport. Blocking reagents and procedures are well known in the art.

Also featured in accordance with the present invention are kits fordiagnosing pathology of the liver or biliary system. In one embodiment,the kits include a reagent that specifically interacts with glycosylmoieties, and instructions for using the kit in a method for diagnosingpathology of the liver. The reagent can be a molecule that directlylabels oligosaccharides such as 2-aminobenzamide, or can be a moleculethat is coupled to a detectable moiety as described herein. In someembodiments, the kits further comprise a secondary reagent thatspecifically recognizes the primary reagent, which is labeled with adetectable moiety. The kits can also include positive and negativecontrols.

In preferred embodiments, the reagent is comprised of antibodies, andany antibody that specifically binds to the glycosyl moiety of interestcan be used in the devices. In more preferred embodiments, the reagentis comprised of proteins that can specifically interact withcarbohydrates. In still more preferred embodiments, the reagent iscomprised of lectins. The preferred glycosyl moiety is fucose.

In some embodiments, the kits further include a solid support toimmobilize the reagent or the analyte from the test sample isolated fromthe patient. The solid support can be any solid support describedherein. The kit may further include coupling agents to facilitateimmobilization of the reagent or analyte to the solid support. In someembodiments, the reagent is provided pre-coupled to the solid support.

The kits can contain materials sufficient for one assay, or can containsufficient materials for multiple assays.

Also featured in accordance with the present invention are methods fordetecting glycosylated proteins in a sample. Such methods comprisingcontacting the sample with a lectin to form a complex betweenglycosylated proteins in the sample and the lectin, and then detectingthe glycosylated protein-lectin complex. In some embodiments, themethods comprise contacting the sample with an antibody to captureglycosylated proteins in the sample. The antibody can be specific for aparticular protein, for a particular glycosyl moiety, or for aparticular protein-glycosyl moiety complex. The antibody can be coupledto a solid support. The sample can then be washed, and the lectinapplied to the washed sample. The lectin can be coupled to a detectablemoiety, and directly detected, or the lectin can be contacted with asecondary reagent coupled to a detectable moiety, e.g., an anti-lectinantibody, and then detected.

As will be appreciated, aspects of the present invention find greatutility in the evaluation of the presence or absence of liverpathologies in humans. It will also be seen by persons of ordinary skillin the art that the present invention may be applied to animals, bothfor prognostic or diagnostic purposes, for the monitoring of therapeuticefficacy or otherwise in manners analogous to those methods useful inhumans. Additionally, however, aspects of the present invention may beused to monitor the progress of animal studies in a researchenvironment. Thus, assessment of glycosylation in circulating or otherproteins in mice, rats, dogs and other animals can inform personsperforming research involving such animals of the pathological status ofthe livers of such animals. In this way, for example, toxicity ofproposed or existing pharmaceuticals, additives, adjuvants, industrialor agricultural chemicals or any of a wide variety of chemicals,biochemical species, environmental or industrial pollutants or othermaterials may be evaluated.

It will be seen that such evaluation of toxicity is useful in, e.g.,drug trials preparatory to regulatory submissions. Additionally, thetoxicity of a host of species may be evaluated in a direct, convenientway. Moreover, it is well known that many extant pharmaceuticals,including statins, antineoplastic agents and others have significantliver toxicity. Toxicity is also a concern when drugs are abused oroverdosed. The present invention allows monitoring of such toxicity inthese circumstances for pre- or post approval pharmaceutical or, indeed,for unlawful drugs.

The following examples are provided to describe the invention in moredetail. They are intended to illustrate, not to limit the invention.

Example 1 General Experimental Procedures

Glycan Analysis.

Serum was obtained from patients clinically diagnosed as infected withchronic HBV, and from patients clinically diagnosed as having HCC,patients clinically diagnosed as having liver cirrhosis, and fromcontrol subjects with no evidence of any liver disease. Serum sampleswere stored at −80° C. until analysis.

Protein aliquots (1 mg/mL) were denatured with 1% SDS, 50 mMβ-mercaptoethanol for 10 min at 100° C. The solution was cooled, andsupplemented with NP-40 to a concentration of 5.75%. PNGase F (ProZyme,San Leandro, Calif.) was added to a final concentration of 1 mU(IUB)/μL,and a cocktail of protease inhibitors was added to the mix. The solutionwas then incubated for 24 hours at 37° C. The oligosaccharides separatedfrom the proteins in the sample were recovered and purified by solidphase extraction using a porous graphite matrix (LudgerClean H, LudgerLimited, Oxford, UK). Free oligosaccharides were labeled with2-aminobenzamide, and purified using commercially available kits (LudgerLimited UK).

Fluorescently-labeled glycans were subsequently analyzed by HPLC using anormal phase column (TSK amide 80 column). The mobile phase consisted ofSolvent A (50 mM ammonium formate, pH4.4) and Solvent B (acetonitrile)and the gradient used was as follows: linear gradient from 20-58%Solvent A at 0.4 mL/minute for 152 min, followed by a linear gradientfrom 58-100% Solvent A for the next 3 min. The flow rate was increasedto 1.0 mL/minute and the column washed in 100% Solvent A for 5 min.Following the wash step, the column was equilibrated in 20% Solvent Afor 22 min in preparation for the next sample run. HPLC analysis wasperformed using the Waters Alliance HPLC System, complemented with aWaters fluorescence detector, and quantified using the MillenniumChromatography Manager (Waters Corporation, Milford, Mass.). Glycanstructures were identified by comparison to known standards and bysequential exoglycosidase digestion.

Lectin Extraction and Analysis.

Immunoglobulins were removed from the samples (media and serum) using aProteinA/G column (Pierce, Rockford, Ill.) prior to the lectinextraction. Samples were supplemented with a lectin binding solution,bringing the final concentration of the sample to 20 mM Tris bufferedsaline (TBS), 1 mM calcium chloride, 1 mM magnesium chloride, and 1 mMmanganese chloride (pH 7.0). The samples were incubated for 16 hours at4° C. with an array of agarose-bound fucose-recognizing lectins. Theselectins consisted of Lens culinaris (LCH), Pisumsativum sativum (PSA)and Vicia faba (VFA) and recognize branched mannoses with the alphafucose determinant (all purchased from EY laboratories, San Mateo,Calif.). Incubation was performed in a microcentrifuge tube prior totransfer to a Costar 0.45 μM Spin-X column (Corning, Acton, Mass.). Thelectin column was washed thoroughly with lectin binding solution beforethe bound fraction was eluted using the appropriate inhibitorymonosaccharides (200 mM methyl-α-D-glucopyranoside, 200 mMα-methyl-D-manno-pyranoside). The bound and unbound fractions werebuffer exchanged into TBS using Milllipore YM-3Centricon devices andsubjected to glycan analysis or 2DE. Protein levels were monitoredthroughout all extractions.

2-Dimensional Gel Electrophoresis.

Samples were diluted in buffer (7M urea, 2M thiourea, 4% CHAPS, 65 mMDTT, 5 mM TBP, and 0.4% ampholytes), vortexed periodically for 1 h, andapplied to an 18 cm pH3-10NL IPG strip (Amersham, Piscataway, N.J.). Gelrehydration was carried out for 14 h at 50 V, and focused using theProtean (Bio-RadLaboratories Headquarters, Hercules, Calif.) IEFapparatus. After focusing, gel strips were reduced in 6 M urea, 2% SDS,1.5% DTT, 30% glycerol, and 50 mM Tris pH 6.8 for 10 min and alkalatedin 6 M urea, 2% SDS, 3% iodoacetamide, 30% glycerol and 50 mM Tris pH6.8. The second dimension was resolved with an 8-18% acrylamide-0.8% PDAgradient gel on a Protean II xi cell (Bio-Rad) with the runningconditions set to 20 mA/gel for 20 min and 40 mA/gel for 4 h with gelscooled to 14° C. Gels were fixed (30% EtOH/5% phosphoric acid) andstained with a colloidal Coomassie brilliant blue stain. For allsamples, gels were run in quadruplicate and only differences that wereconsistent in all gels were considered meaningful.

Gel Imaging and Analysis.

Gels were digitally imaged using a 16-bit cooled CCD camera (FluorChem8000, Alpha Innotech, San Leandro, Calif.). TIFF files of the gel imageswere analyzed using NonLinear Dynamics Progenesis Workstation gelimaging software package (Nonlinear USA Inc., Durham, N.C.). Thepolypeptide features in each gel image were delineated and then thesoftware determined the total intensity of pixels within each feature(the integrated intensity). Polypeptide features were normalized byusing the integrated intensity of each feature and expressing it as apercent of the sum of integrated intensities of the entire gel.

Mass Spectrometry.

MALDI Analysis. Protein spots were excised from colloidal Coomassie bluestained gels, de-stained, and digested with trypsin. Recovered peptideswere concentrated and desalted using Zip Tip C18 (Millipore, Bedford,Mass.) according to manufacturer's directions and prepared for MALDI-TOFmass spectrometry by mixing 0.5 μL of peptide mixture with 0.5 μL of 10mg/mL of alpha-cyano 4-hydroxy cinnamic acid, 1% formic acid in 50%acetonitrile and allowing the droplet to dry on the MALDI plate. Peptidemass maps were obtained using a Voyager-DE ProMass Spectrometer (PEBiosystems, Foster City, Calif.) operated in positive ion reflectronmode. Proteins were identified from the peptide mass maps using theMASCOT online database www.matrixscience.com to search the nonredundantprotein database.

LC MS/MS Analysis.

Peptide identification was performed on a ThermoFinnigan LCQ ion trapmass spectrometer (ThermoElectron Corporation, CA) equipped with on-linemicrocapillary HPLC (Eldex, Napa, Calif.) and microspray ionizationsource that was built in-house. The microspray consists of amicrocapillary column, picotip 360×75 μm, with an integrated 15 μm tip(New objective, Woburn Mass.) self-packed with Reliasil C18 resin(Column Engineering, Ontario, Calif.) to a length of 10 cm. An aliquotof the sample was loaded via a pressure bomb into a capillary sampletrap (Upchurch, Wash.) self-packed with C18, then placed just upstreamof the microcapillary column. The HPLC is programmed to produce a 3 hgradient (5%-65% B) at30 μL/min. Prior to the trap, passive flowsplitting is used to reduce the flow down to ˜500 n1/minute. Buffer Aconsists of 5% acetonitrile+1% acetic acid; buffer B consists of 90%acetronitrile+1% acetic acid. The LCQ was programmed to perform a fullscan from 450 to 2000 m/z, followed by 3 data dependent MS/MS scans setto pick the most abundant ion species from the full scan.

Data Analysis and Interpretation.

Mass spectrometry data (spectra) was searched against the nonredundanthuman database using SeQuest (Thermo Electron Corporation). Peptidesthat score above the threshold value of 1.5 XCorr (singly)/2.0 XCorr(doubly)/2.5 XCorr (triply) were then manually verified. In all cases,the bound peptides should contain the N-linked glycosylation sequon,N—X—S/T that has been converted to a D-X-S/T by the action of the PNGaseF enzyme. This results in a Mass difference of 0.9840 Da as compared tothe glycosylated sequence.

Immunoblotting.

Equal volumes of patient sera from either total serum (0.5 pt/lane), nonlectin bound (unbound) or lectin bound (bound) were resolved by SDS-PAGEon 4-20% polyacrylamide gradient gels. The proteins were transferred toa PVDF membrane by immunoblotting. The membranes were blocked byincubating with a blocking buffer of 1×TBS (50 mM Tris-HCl, pH 7.6, 150mM sodium chloride) made 5% nonfat dried milk, and 0.1% Tween 20 for 1 hat room temperature. The blots were then incubated overnight withdesired antibody and developed using chemiluminescent detection system(“ECL Plus,” Amersham Pharmacia Biotech, Arlington Heights, Ill.). Blotswere visualized using an Alphalnnotech FluorChem CCD camera withAlphaEase spot densitometry software (Alphalnnotech Corp., San Leandro,Calif.).

Example 2 Core Fucosylation is Increased in Hepatocellular Carcinoma

A targeted glycoproteomic methodology that allows for the identificationof glycoprotein biomarkers in serum was developed. The methodology firstidentifies changes in N-linked glycosylation that occur with thedisease. These changes act as a tag so that specific proteins thatcontain that glycan structure can be extracted. Initial experiments inan animal model revealed a protein, GP73, that is more sensitive atdetecting HCC than currently used markers such as AFP. In the animalmodel of HCC, the change in glycosylation was an increase in corefucosylation (Block et al. 2005). This change was also observed inpeople who developed HCC. An example of the increase in fucosylation isshown in FIG. 2. This sample set consisted of 16 samples from 8 patientsat two time points: before the development of cancer, and after thediagnosis of cancer. In this case, all patients classified as beforewere clinically diagnosed with cirrhosis. Samples classified as afterwere clinically diagnosed with HCC by ultrasound and biopsy. As thisfigure shows, patients before the diagnosis of HCC have much lowerlevels of the core fucosylated bi-antennary glycan (FcA2G2) as comparedto after the diagnosis of HCC. For example, patient 121 had 7.23% of theFcA2G2 glycan before the diagnosis of HCC and 13.2% after the diagnosisof HCC. As seen in FIG. 2B, this upward trend was true of all patientsexamined. These results highlight the fact that increased levels ofα-1,6 linked core fucosylation are associated with the development ofHCC. T his is true in samples that were AFP positive (>20 ng/ML) or AFPnegative. That is, as shown in FIG. 2B, patients 965, 370, 842, 999 and978 were all AFP negative, yet had increased levels of the FcA2G2glycan. For all the patients examined so far those who are normal, haveactive hepatitis, or have cirrhosis had an average of 8.44%+/−0.75(n=19) of the core fucosylated glycan, while those patients diagnosedwith HCC had an average of 12.5%+/−1.83 (n=17) of the core fucosylatedglycan. This difference was statistically different with a P value of0.001.

Example 3 Identification of Fucosylated Glycoproteins in HepatocellularCarcinoma

As the level of fucosylation was increased in patients with HCC, it wasimperative to determine those proteins that had increased fucosylation.Therefore, the fucosylated glycoproteins associated with sera fromeither pooled normal or pooled HCC positive individuals were extractedusing lectins and the proteome analyzed by either two dimensional gelelectrophoresis (2DE) or by a simple LC MS/MS based methodology. Usingthese methodologies it was observed that glycoproteins such asfucosylated (Fc) α-1-acid glycoprotein, Fc-ceruloplasmin,Fc-α-2-macroglobulin, Fc-hemopexin, Fc-Apo-D, Fc-HBsAg, and Fc-Kininogenincreased in patients with HCC, while the levels of Fc-haptoglobindecrease in the those patients (Table 1). Interestingly, severalimmunoglobulin molecules (IgG, IgA and IgM) were also found in thefucosylated proteome of those patients with cancer. However, furtheranalysis has shown that these proteins become fucosylated primarily incirrhosis and not just cancer (FIG. 4).

TABLE 1 Proteins identified as altered in patients with HCC. Fc-GP-73Fc-Hemopexin Fc-HBsAg Fc-hepatitis B viral partideFc-alpha-acid-glycoprotein Fc-alpha-1-antichymotrypsin (includingFc-Alpha-1-antichymotrypsin His-Pro-less) Fc-alpha-1-antitrypsinFc-Serotransferrin Fc-Ceruloplasmin Fc-alpha-2-macroglobulinFc-alpha-2-HS-glycoprotein (Fetuin A) Fc-Haptoglobin Fc-Fibrinogen gammachain precursor Fc-IgG Fc-IgA Fc-APO-D Fc-IgM Fc-Kininogen Fc-Histidinerich glycoprotein Fc-Complement factor 1 precursor (includingFc-Complement factor I heavy chain Fc-Complement factor I light chain)Fc-Complement C1s component Fc-Complement factor B precursor (includingComplement factor B Ba fragment and Fc-Complement factor B Bb fragment)Fc-Complement C3 precursor (including Fc-Complement C3 beta chainFc-Complement C3 alpha chain Fc-C3a anaphylatoxin Fc-Complement C3balpha′ chain Fc-Complement C3c fragment Fc-Complement C3dg fragmentFc-Complement C3g fragment Fc-Complement C3d fragment and Fc-ComplementC3f fragment) Fc-Complement C5 (including Fc-Complement C5 beta chainFc-Complement C5 alpha chain Fc-C5a anaphylatoxin Fc-Complement C5alpha′ chain) Fc-Complement C7 Fc-alpha-1 B glycoproteinFc-B-2-glycoprotein (apo H) Fc-Vitamin D-binding proteinFc-Inter-alpha-trypsin inhibitor heavy chain H2 Alpha-1 B-glycoproteinFc-Angiotensinogen precursor Fc-Angiotensin-1 Fc-Angiotensin-2Fc-Angiotensin-3 Fc-GARP protein Fc-beta-2-glycoprotein Fc-Clusterin(Apo J) Integrin alpha-8 precursor glycoprotein (including Fc-Integrinalpha-8 heavy chain and Fc-Integrin alpha-8 light chain Fc-hepatitis Cviral partide Fc-Leucine-rich repeat-containing protein 32 precursor.

Example 4 Levels of Fc-GP73 and Fc-Hemopexin are Increased in Patientswith Hepatitis B-Induced Hepatocellular Carcinoma

The level of Fc-GP73 and Fc-hemopexin were examined in a small blindedpatient cohort (from our collaborator Dr. Chau-Ting Yeh, Director,Digestive core lab and Hepatoma research team, Liver Research Unit,Chang Gung Memorial Hosp, Taiwan) containing a total of 80 patients withvarying degrees of liver disease (Table 2). Analysis of these sampleswas performed for total GP73 level, for the level of fucosylated GP-73(Fc-GP73), and for the level of fucosylated hemopexin (Fc-hemopexin).Total GP73 was analyzed by immunoblot using whole serum. Fucosylatedspecies were analyzed by lectin extraction of 5 μl of serum (LCH)followed by immunoblotting of the fucosylated fraction and imaging usingan Alphalnnotech FluorChem CCD camera with AlphaEase spot densitometrysoftware. The sensitivity, specificity, and positive predicative valuesfor these three markers are presented in Table 3. As this figure shows,using a cut-off of 5 fold greater then purchased healthy serum (SigmaChemical Co., St. Louis, Mo.) total GP73 had a sensitivity of 65% and aspecificity of 90%, which was very similar to our larger blinded studyusing this marker (Marrero et al. (2005) J. Hepatol. 43:1007-12); 69%sensitivity and 86% specificity). Even more promising then total GP73were Fc-GP73 and Fc-hemopexin. Fc-GP73 had a sensitivity of 90% and aspecificity of 100% (using a cut-off of 100 fold greater then purchasedhealthy serum (Sigma Chemical Co), while Fc-hemopexin had a sensitivityof 95% and a specificity of 100% (using a cut-off of 20 fold greaterthen purchased healthy serum (Sigma Chemical Co.).

TABLE 2 Samples utilized to compare the predictive value of markers inHBV induced liver disease. Group Clinical Status* Number Age Range 1Healthy Subjects 20 21-64 2 HBV+, inactive (carrier) 20 26-70 3 HBV+,active (Cirrhosis) 20 22-68 4 HBV infected, HCC 20 25-81 Sigma SerumCommercially available serum 1 unknown *clinical status was determinedby lack of HBsAg (group 1), presence of HBsAg (group 2), presence ofHBsAg along with biopsy to confirm cirrhosis (group 3), or presence ofHBsAg along with the diagnosis of HCC by MRI (group 4)

TABLE 3 Sensitivity, specificity, and positive predictive value of totalGP73, Fc-GP73, and Fc-hemopexin in the patients shown in Table 2. MarkerSensitivity Specificity PPV Total GP73 65%  90%  77% Fc-GP73* 90% 100%100% Fc-Hemopexin* 95% 100% 100% *Analysis of Fc-GP73 and Fc-hemopexinwas performed via immunoblot following Ig-depletion and lectinextraction.

Example 5 Lectin ELISA for Fc-AFP and Fc-Kininogen

The analysis of fucosylated GP73 and fucosylated hemopexin in Table 3was performed by immunoblot of the fucosylated proteome. This requiredthe removal of immunoglobulin and lectin extraction of each sample. Asthis is quite laborious, a lectin-ELISA to allow for much higherthroughput was developed (see FIG. 3). Initial experiments focused onthe development of a lectin-ELISA for Fc-AFP. AFP is known to befucosylated and several reports in the literature were available toallow for comparison (Naitoh et al. (1999) J. Gastroenterol. Hepatol.14:436-45). For assay development, a selected sample set of 60 patientswas utilized as previously described (Marrero et al. (2005) J. Hepatol.43:1007-12). 20 patients were infected with HCV but otherwise consideredhealthy, 20 patients were infected with HCV and diagnosed withcirrhosis, and 20 patients were infected with HCV and diagnosed withHCC. The samples in this set were chosen to include many AFP+individuals. In addition, human serum (Sigma Chemical Co.) was utilizedas a control to which all other patients groups were compared. Briefly,50 μl of human serum is diluted in 150 μl of lectin-binding buffer (20mM Tris, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MoCl2 chloride, pH 7.0) andadded directly to the Heterophilic Blocking Tubes prior to addition toplates for 2 hours at 370 C. Subsequently, plates were washed 10 timeswith 2% Tween 20 in lectin binding buffer before lectin was added.Fucosylated glycoforms were detected with a biotin labeled LensCulinaris or Aleuria aurantia lectin (Vector Laboratories, Burlingame,Calif.). Lectin was detected using a fluorescein labeled streptavidinsystem and subsequently, samples were measured in the Cytofluor 4000Fluorescence Plate Reader (MTX Lab Systems, Inc. Vienna, Va.) at theappropriate wavelength. The relative fluorescent intensity in eachsample was compared to that observed in purchased normal serum. Table 4shows the results of the analysis with Fc-AFP. In this sample set, usinga cut-off of 5 fold greater then purchased healthy serum (Sigma ChemicalCo.), Fc-AFP had a sensitivity of 70%, a specificity of 90% and apositive predicative value (PPV) of 91%. This is consistent with whathas been reported with Fc-AFP (Naitoh et al. (1999) J. Gastroenterol.Hepatol. 14:436-45). In this same sample set, total AFP had asensitivity of 70%, a specificity of 70%, and a PPV of 77% (using acut-off of 20 ng/ml). Using the same sample set, Fc-kininogen wasanalyzed using the same methodology as for AFP. Fc-Kininogen wasidentified in proteomic analysis as being present in the cancer samplesand utilized initially as the antibody was easily available. As Table 4shows, Fc-kininogen had a sensitivity of 80%, a specificity of 95% and aPPV of 95% (using a cut-off of 3 fold greater then purchased healthyserum (Sigma Chemical Co).

TABLE 4 Sensitivity, specificity, and PPV for total AFP, fucosylatedAFP, and fucosylated kininogen as determined by Lectin-ELISA. MarkerSensitivity Specificity PPV AFP 70% 70% 77% Fc-AFP 70% 90% 91%Fc-Kininogen 80% 95% 95%

Example 6 Human Immunoglobulins Become Fucosylated with the Developmentof Cirrhosis

As shown in Table 1, over 50 glycoproteins that have alteredglycosylation as a function of liver disease have been identified.Although the increase in many of these fucosylated glycoproteinscorrelates with the development of hepatocellular carcinoma (HCC), someof these glycoproteins were found to alter with other liver diseases aswell. For example, it was observed that human Immunoglobulins becomehyper-fucosylated with the development of liver cirrhosis. Since IgG wasdetermined to be the major source of fucosylated protein in the serum ofthose with cirrhosis, in the initial small set of samples, it wasreasoned that the level of fucosylated IgG might correlate with adiagnosis of cirrhosis. Moreover, based on the results shown in FIG. 3and Table 4, it seemed that a simple lectin based assay could bedeveloped to detect fucosylated IgG.

Briefly, fucosylated IgG was detected by incubating 5 μl of serum withwells coated with periodate oxidized mouse anti-human IgG for 2 hours.Subsequently fucosylated human IgG was detected by incubation withbiotinylated Aleuria aurantia lectin (AAL). Bound lectin was detectedusing RDye™ 800 Conjugated STREPTAVIDIN and signal intensity measuredusing the Odyssey® Infrared Imaging System (LI-COR Biotechnology,Lincoln, Nebr.). In all cases sample intensity was compared tocommercially purchased human serum (Sigma Chemical Co.).

Using this assay, the relative amount of fucosylated IgG was determinedin more than 200 serum samples from with normal patients, or those withvarying degrees of liver disease. In addition, patients with non-liverdiseases were examined. It is important to note that the operator of thefucosylated IgG detection assay was not aware of the diagnosisassociated with serum sample, at the time of the testing. The resultsare shown in FIG. 4.

The results are expressed as fold increase over sigma. The signaldetected in the samples from the 70 healthy subjects (no evidence ofHBV, HCV or liver disease) was consistently low, less than 2 fold ofthat observed in commercially purchased serum. In contrast, all of thecirrhosis patients had values greater than 2 fold above commerciallypurchased serum. The mean level of increase was 13-fold abovecommercially purchased serum. HCV infected patients with varying levelsof fibrosis also had elevated levels of fucosylated IgG. Interestingly,the level of fucosylated IgG varied in these patients based upon thelevel of fibrosis. Those patients with stage 0-2 fibrosis had a mean 3fold increase in fucosylated IgG while those patients with stage 4 orgreater fibrosis had a mean 13 fold increase in fucosylated IgG. Using acut-off of 5 fold above commercially purchased human serum, this assayhad a sensitivity of 92%, a specificity of 96% at differentiating stage1-2 fibrosis from stage 3-6 fibrosis/cirrhosis. The positive predictivevalue was 92% and the negative predictive value was 96%.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

What is claimed:
 1. A method for diagnosing a liver pathology in asubject suspected of having the liver pathology comprising: obtainingbiological fluid from the subject; contacting said biological fluid witha fucose-binding lectin and allowing said lectin to bind to a protein insaid biological fluid, wherein said protein is selected from Hemopexin,HBsAg, hepatitis B viral particle, alpha-1-antichymotrypsin,alpha-1-antichymotrypsin His-Pro-less, Ceruloplasmin,alpha-2-macroglobulin, alpha-2-HS-glycoprotein, Haptoglobin, Fibrinogengamma chain precursor, immunoglobulin, APO-D, Kininogen, Histidine richglycoprotein, complement factor 1 precursor, complement factor I heavychain, complement factor I light chain, Complement C1s, Complementfactor B precursor, complement factor B Ba fragment, Complement factor BBb fragment, Complement C3 precursor, Complement C3 beta chain,Complement C3 alpha chain, C3a anaphylatoxin, Complement, C3b alpha′chain, Complement C3c fragment, Complement C3dg fragment, Complement C3gfragment, Complement C3d fragment, Complement C3f fragment, ComplementC5, Complement C5 beta chain, Complement C5 alpha chain, C5aanaphylatoxin, Complement C5 alpha′ chain, Complement C7, alpha-1 Bglycoprotein, B-2-glycoprotein, Vitamin D-binding protein,Inter-alpha-trypsin inhibitor heavy chain H2, Alpha-1B-glycoprotein,Angiotensinogen precursor, Angiotensin-1, Angiotensin-2, Angiotensin-3,GARP protein, beta-2-glycoprotein, Clusterin (Apo J), Integrin alpha-8precursor glycoprotein, Integrin alpha-8 heavy chain, Integrin alpha-8light chain, hepatitis C viral particle, and Leucine-richrepeat-containing protein 32 precursor; quantifiably detecting boundlectin in the biological fluid to obtain a detected bound lectin value;and comparing the detected bound lectin value with a reference value forlectin binding on said protein in a comparable biological fluid ofsubjects without said liver pathology, said detected bound lectin valuerelative to the reference value being indicative of the presence orabsence of said liver pathology, wherein a detected bound lectin valuethat deviates from said reference value to a statistically significantdegree is indicative of the presence of said liver pathology, orcomparing the detected bound lectin value with a reference value forlectin binding on said protein in a comparable biological fluid ofsubjects in which said liver pathology is known to be present, saiddetected bound lectin value relative to the reference value beingindicative of the presence or absence of said liver pathology, wherein adetected bound lectin value that deviates from said reference value to astatistically significant degree is indicative of the absence of saidliver pathology.
 2. The method according to claim 1 wherein said liverpathology is cirrhosis.
 3. The method according to claim 1 wherein saidliver pathology is fibrosis.
 4. The method according to claim 1 whereinsaid liver pathology is hepatocellular carcinoma.
 5. The methodaccording to claim 1 wherein said liver pathology is hepatitis.
 6. Themethod of claim 1 wherein the biological fluid is whole blood, serum,urine, saliva, tears, or mucous.
 7. The method of claim 1, wherein thelectin is coupled to a detectable moiety.